Dye transfer inhibition

ABSTRACT

The transfer of a textile dye from a dyed fabric to another fabric during washing or rinsing is inhibited by adding an enzyme exhibiting peroxidase activity or an enzyme exhibiting a suitable oxidase activity to the wash liquor in which said fabrics are washed and/or rinsed. Peroxidase is produced extracellularly by some strains of Bacillus pumilus. The novel peroxidase preparation from B. pumilus is a microperoxidase, i.e. it contains hemopeptide as an active component. The preparation has improved stability at high temperature, at high pH and at high concentrations of hydrogen peroxide. It can be produced without undesired catalase activity.

CROSS-REFERENCE TO RELATED APPLICATION

This is a divisional application of application Ser. No. 08/105,222,filed Aug. 11, 1993, now U.S. Pat. No. 5,605,832 which is a divisionalof Ser. No. 07/599,331 filed Oct. 17, 1990 now U.S. Pat. No. 5,273,896,which is a continuation-in-part of Ser. No. 07/421,414 filed Oct. 13,1989 now abandoned. The contents of Ser. No. 08/105,222 are incorporatedherein by reference in their entirety.

FIELD OF INVENTION

The present invention relates to an enzymatic process for inhibiting thetransfer of dye from a dyed fabric to another fabric during washing, toa bleaching agent for use in the process, and to a process for bleachingdyes in solution. This invention also relates to a hemopeptide withperoxidase activity, to a peroxidase preparation, and to production anduse of such a preparation.

BACKGROUND OF THE INVENTION

The use of bleaching agents in washing procedures and as constituents ofdetergent compositions is well known in the art. Thus, bleaching agentsare incorporated in or sold as constituents of a major part of thecommercially available detergent compositions. Important conventionalbleaching agents incorporated in detergent compositions are compoundswhich act as precursors of hydrogen peroxide formed in the course of thewashing procedure. Perborates and percarbonates are the most importantexamples of compounds which are employed as bleaching agents and whichexert a bleaching effect in this fashion. The detailed mechanism ofbleaching by means of these bleaching agents is not known at present,but it is generally assumed that the hydrogen peroxide formed duringwashing converts coloured substances (responsible for stains on fabric)into noncoloured materials by oxidation and that some oxidation of thecoloured substances may also take place due to their direct interactionwith perborate or percarbonate.

One drawback of these commonly used bleaching agents is that they arenot particularly efficient at the lower temperatures at which colouredfabrics are usually washed. Their efficiency may be enhanced by the useof activators (e.g. organic acid anhydrides, esters or imides) whichgive rise to the formation of peracids.

Apart from being employed for bleaching stains on fabric, suchconventional bleaching agents have also been suggested for preventingsurplus dyes from coloured fabrics which leach from the fabrics whenthese are washed from being deposited on other fabrics present in thesame wash (this phenomenon is commonly known as dye transfer). Theproblem of dye transfer, of course, is most noticeable when white orlight-coloured fabrics are washed together with fabrics of a darkercolour from which dye is leached during washing.

It has, however, been found that the currently employed bleachingagents, whether activated or not, are not particularly effective ininhibiting dye transfer, possibly because the rate at which they oxidizedissolved dyes is rather slow. On the other hand, peracids formed fromthe bleaching activators are active against dyes on fabric so as tocause discolouration of the fabric in question.

U.S. Pat. No. 4,077,768 discloses the use of iron porphin, haeminchloride or iron phthalocyanine, or derivatives thereof together withhydrogen peroxide for dye transfer inhibition. It is indicated thatthese compounds act as catalysts for the bleaching process whereby theyprovide an increase in the rate at which dissolved dyes are oxidised(or, in other words, bleached) without causing any discolouration of thedye in the fabric. However, these catalysts are destroyed by thepresence of excess hydrogen peroxide which makes it necessary to controlthe release of hydrogen peroxide so that only the quantity of hydrogenperoxide needed to effect the inhibition of dye transfer should bepresent in the wash water at any time. Such controlled release of thebleaching agent may be difficult to achieve.

Peroxidase activity catalyses oxidation of a substrate (an electron orhydrogen donor such as lignin) with hydrogen peroxide.

High-molecular peroxidases (E.C. 1.11.1.7) are produced intracellularlyby some microorganisms. Thus, S. Loprasert et al., Journal of GeneralMicrobiology (1983), 134, 1971-1976 describe a peroxidase-catalase fromBacillus stearothermophilus with molecular weight 175,000 and U.S. Pat.No. 4,698,306 describes a peroxidase from Coprinus with molecular weight37,000-41,000. Also, so-called microperoxidases of mammalian origin areknown; these are hemopeptides, typically with molecular weight in therange 1,500-3,000, exhibiting peroxidase activity (e.g. DE 3134526).

Use of peroxidase together with hydrogen peroxide or a hydrogen peroxideprecursor has been suggested e.g. in bleaching of pulp for paperproduction (SE 88/0673), in treatment of waste water from pulpproduction (U.S. Pat. No. 4,623,465, JP-A 2-31887) and for improvedbleaching in laundry detergents (WO 89/09813). Pending U.S. patentapplication Ser. No. 07/421,414 (filed Oct. 13, 1989) and a co-pendingPCT application disclose the use for dye transfer inhibition duringlaundering.

It is the object of the invention to provide an improved peroxidase forthese purposes. The peroxidase should be active and stable at hightemperature and H₂ O₂ concentration, especially at alkaline conditions.It should be free of catalase as this activity breaks down hydrogenperoxide that is needed in the reaction. For better production economy,the peroxidase should be microbial and should be producedextracellularly by the microorganism in question.

SUMMARY OF THE INVENTION

It has surprisingly been found possible to bleach coloured substancesleached from dyed textiles or from textiles soiled with a colourant in asolution of wash liquor thereby preventing the coloured substance inquestion from being deposited on other textiles in the wash liquor, whenenzymes utilizing hydrogen peroxide or molecular oxygen for theoxidation of organic or inorganic substances, including colouredsubstances, are added to the wash liquor. Such enzymes are usuallytermed peroxidases and oxidases, respectively.

Accordingly, the present invention relates to a process for inhibitingthe transfer of a textile dye from a dyed fabric to another fabric whensaid fabrics are washed and/or rinsed together in a wash liquor, theprocess comprising adding an enzyme exhibiting peroxidase activity or anenzyme exhibiting a suitable oxidase activity to the wash liquor inwhich said fabrics are washed and/or rinsed. In the present context, theterm "enzyme exhibiting peroxidase activity" is understood to indicatean enzyme with a mode of action similar to that of a peroxidase and willbe used synonymously therewith. Similarly, the term "enzyme exhibiting asuitable oxidase activity" is understood to indicate an enzyme with asimilar mode of action to that of an oxidase and is meant to besynonymous therewith in the following. Suitable oxidases include thosewhich act on aromatic compounds such as phenols and related substances.

One or more substrates for the enzyme may also be added at the beginningof or during the washing and/or rinsing process, in particular when theenzyme is one with peroxidase activity as, in the case of oxidases,molecular oxygen is usually present in sufficient quantities. When theenzyme used in the process of the invention is a peroxidase, hydrogenperoxide or a precursor of hydrogen peroxide, preferably perborate orpercarbonate, will therefore typically be added as the substrate.

It is well recognized in the art (cf. for instance B. C. Saunders etal., Peroxidase, London, 1964, p. 10 ff.) that peroxidases act onvarious amino and phenolic compounds resulting in the production of acolour. In view of this, it must be considered surprising thatperoxidases (and certain oxidases) may also exert an effect on colouredsubstances in solution such that dye transfer is inhibited. While themechanism governing the ability of these enzymes to effect dye transferinhibition has not yet been elucidated, it is currently believed thatthe enzymes act by reducing hydrogen peroxide or molecular oxygen andoxidizing the coloured substance (donor substrate) dissolved ordispersed in the wash liquor, thereby either generating a colourlesssubstance or providing a substance which is not adsorbed to the fabric.This reaction is shown in Reaction Scheme 1 below (for peroxidases) andReaction Scheme 2 below (for oxidases useful for the present purpose).

Reaction Scheme 1:

Donor substrate+H₂ O₂ →oxidized donor+2 H₂ O

Reaction Scheme 2:

Donor substrate+O₂ →oxidized donor+2 H₂ O

It has previously been reported that peroxidases may decolourize certainpigments (cf. for instance W. Schreiber, Biochem. Biphys. Res. Commun.63 (2), 1975, pp. 509-514, describing the degradation of3-hydroxyflavone by horseradish peroxidase; A. Ben Aziz, Phytochemistry10, 1971, pp. 1445-1452, describing the bleaching of carotene by meansof a peroxidase; and B. P. Wasserman, J. Food Sci. 49, 1984, pp. 53614538, describing the decolourization of betalain by horseradishperoxidase). Ben Aziz et al. and Wasserman et al. present the bleachingaction of peroxidases on carotene and betalain, respectively, as aproblem when using these pigments as food colourants, which problem mustbe counteracted by including an anti-oxidant in the foodstuff inquestion. Thus, they do not consider the peroxidase-mediated bleachingof these pigments to have any practical utility in itself.

Although these publications describe test methods whereby the respectivepigments are incubated with the enzyme in solution, the pigments inquestion are all pure compounds of natural origin and are also readilybleached by the bleaching agents usually incorporated in moderndetergents (cf. for instance Second World Conference on Detergents, A.R. Baldwin (ed.), American Oil Chemist's Society, 1978, pp. 177-180).

Contrary to this, the commonly used textile dyes, when dissolved ordispersed in wash liquors, are generally resistant to oxidation byatmospheric oxygen and also, to a greater or lesser extent, to thebleaching agents currently used in detergents which, as noted in U.S.Pat. No. 4,077,768, are inefficient dye transfer inhibitors as they acttoo slowly on the dispersed or dissolved dyes. Under thesecircumstances, it must be considered surprising that the enzymes used inthe present process are, in fact, able to oxidize these dyes. Othercommonly used bleaching agents which may have an effect on textile dyesin solution or dispersion, e.g. hypochlorite, also attack dye on or inthe fabrics, resulting in discolouration thereof. It is an importantadvantage of the enzymes used in the process of the invention that theydo not cause any appreciable colour degradation in the dyed fabricitself. A comprehensive catalogue of commonly used textile dyes, bothsynthetic (such as azo dyes) and natural or nature-identical (by whichis meant a substance which is produced synthetically, but which instructure and properties is identical to the natural compound), e.g.indigo, is found in the Color Index, 3rd ed. Vol. 1-8.

In another aspect, the present invention relates to a process forbleaching textile dyes in solution or dispersion, the process comprisingadding an enzyme exhibiting peroxidase activity or an enzyme exhibitinga suitable oxidase activity to said solution or dispersion. It iscontemplated that, apart from having utility in inhibiting dye transferduring a washing or rinsing process, the ability of these enzymes tobleach dyes in solution may also make them useful for treating wastewater from the textile industry forming part of a waste disposalprocess.

In a further aspect, the present invention relates to a bleaching agentfor inhibiting the transfer of a textile dye from a dyed fabric toanother fabric when said fabrics are washed and/or rinsed together, theagent comprising an enzyme exhibiting peroxidase activity or an enzymeexhibiting a suitable oxidase activity. Apart from this utility, thebleaching agent may also be employed in the treatment of waste waterfrom the textile and possibly also other industries, as indicated above.

We have also, surprisingly, discovered that peroxidase is producedextracellularly by some strains of Bacillus pumilus. The novelperoxidase preparation from B. pumilus is a microperoxidase preparation,i.e. it contains hemopeptide as an active component. The preparation hasimproved stability at high temperature, at high pH and at highconcentrations of hydrogen peroxide. It can be produced withoutundesired catalase activity.

Accordingly, the invention provides a hemopeptide characterized by theamino acid sequence SEQ ID NO:3 having R linked to cysteine in position1 and R' linked to histidine in position 5, wherein the sulphur atoms ofthe two cysteines in positions 1 and 4 are linked to the vinyl groups ofheme, and R and R' each represents a peptide chain of 0-10 amino acids.

The invention also provides a peroxidase preparation, characterized bycomprising as an active component a hemopeptide as defined above or onethat contains a heme group linked to a peptide chain containing theamino acid sequence SEQ ID NO:4, wherein each Xaa represents any aminoacid. In another embodiment the invention provides a peroxidasepreparation, characterized by comprising one or more active componentsderived from B. pumilus with molecular weight in the range 800-2500,said preparation being further characterized by a pH optimum in therange 7.5-9, by retaining at least 50% residual activity after 2 hoursat 80° C., and by having optimum activity at H₂ O₂ concentration above20 mM.

The invention further provides a method of producing a peroxidasepreparation, characterized by comprising cultivation of aperoxidase-producing strain of B. pumilus followed by recovery ofperoxidase.

Finally, the invention provides use of the above peroxidase preparationtogether with hydrogen peroxide (optionally formed in situ) in bleachingof lignin-containing material.

DETAILED DESCRIPTION OF THE INVENTION

Examples of suitable oxidases which act on aromatic compounds, inparticular phenolic, e.g. polyphenolic, are catechol oxidase (EC1.10.3.1) or laccase (EC 1.10.3.2). For the sake of convenience, suchoxidases, and peroxidases are collectively termed bleaching enzymes inthe following.

Bleaching enzymes which may be employed for the present purpose may beisolated from and are producible by plants (e.g. horseradish peroxidase)or microorganisms such as fungi or bacteria. Some preferred fungiinclude strains belonging to the subdivision Deuteromycotina, classHyphomycetes, e.g. Fusarium, Humicola, Tricoderma, Myrothecium,Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia,Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM 2672),Humicola insolens, Trichoderma resii, Myrothecium verrucana (IFO 6113),Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERMP-7754), Caldariomyces fumago, Ulocladium chartatum, Embellisia alli orDreschlera halodes.

Other preferred fungi include strains belonging to the subdivisionBasidiomycotina, class Basidiomycetes, e.g. Coprinus, Phanerochaete,Coriolus or Trametes, in particular Corpinus cinereus f. microsporus(IFO 8371), Coprinus macrorhizus, Pahnerochaete chyrsosporium (e.g.NA-12) or Coriolus versicolor (e.g. PR 4 28-A).

Further preferred fungi include strains belonging to the subdivisionZygomycotina, class Mycoraceae, e.g. Rhizopus or Mucor, in particularMucor hiemalls.

Some preferred bacteria include strains of the order Actinomycetales,e.g. Streptomyces spheroides (ATTC 23965), Streptomyces thermoviolaceus(IFO 12382) or Streptoverticilum verticillium ssp. verticillium.

Other preferred bacteria include Bacillus pumillus (ATCC 12905),Bacillus stearothermophilus, Rhodobacter sphaeroides, Rhodomonaspalustri, Streptococcus lactis, Pseudomonas purrocinia (ATCC 15958) orPseudomonas fluorescens (NRRL B-11).

Other potential sources of useful bleaching enzymes (in particularperoxidases) are listed in B. C. Saunders et al., op. cit., pp. 41-43.

Methods of producing enzymes to be used according to the invention aredescribed in the art, cf. for example FEBS Letters 1625, 173(1), Appliedand Environmental Microbiology, Feb. 1985, pp. 273-278, AppliedMicrobiol. Biotechnol. 26, 1987, pp. 158-163, Biotechnology Letters9(5), 1987, pp. 357-360, Nature 326, Ar. 2, 1987 FEBS Letters 4270,209(2), p. 321, EP 179 486, EP 200 565, GB 2 167 421, EP 171 074, andAgric. Biol. Chem. 50(1), 1986, p. 247.

Particularly preferred bleaching enzymes are those which are active atthe typical pH of washing liquors, i.e. at a pH of 6.5-10.5, preferably6.5-9.5, and most preferably 7.5-9.5. Such enzymes may be isolated byscreening for the relevant enzyme production by alkalophilicmicroorganisms, e.g. using the ABTS assay described in R. E. Childs andW. G. Bardsley, Biochem. J. 145, 1975, pp. 93-103.

Other preferred bleaching enzymes are those which exhibit a goodthermostability as well as a good stability towards commonly useddetergent components such as non-ionic, cationic, or anionicsurfactants, detergent builders, phosphate etc.

Another group of useful bleaching enzymes are haloperoxidases, such aschloro- and bromoperoxidases.

The bleaching enzyme may furthermore be one which is producible by amethod comprising cultivating a host cell transformed with a recombinantDNA vector which carries a DNA sequence encoding said enzyme as well asDNA sequences encoding functions permitting the expression of the DNAsequence encoding the enzyme, in a culture medium under conditionspermitting the expression of the enzyme and recovering the enzyme fromthe culture.

A DNA fragment encoding the enzyme may, for instance, be isolated byestablishing a cDNA or genomic library of a microorganism producing theenzyme of interest, such as one of the organisms mentioned above, andscreening for positive clones by conventional procedures such as byhybridization to oligonucleotide probes synthesized on the basis of thefull or partial amino acid sequence of the enzyme, or by selecting forclones expressing the appropriate enzyme activity, or by selecting forclones producing a protein which is reactive with an antibody againstthe native enzyme.

Once selected, the DNA sequence may be inserted into a suitablereplicable expression vector comprising appropriate promotor, operatorand terminator sequences permitting the enzyme to be expressed in aparticular host organism, as well as an origin of replication enablingthe vector to replicate in the host organism in question.

The resulting expression vector may then be transformed into a suitablehost cell, such as a fungal cell, preferred examples of which are aspecies of Aspergillus, most preferably Aspergillus oryzae orAspergillus niger. Fungal cells may be transformed by a processinvolving protoplast formation and transformation of the protoplastsfollowed by regeneration of the cell wall in a manner known per se. Theuse of Aspergillus as a host microorganism is described in EP 238,023(of Novo Industri A/S), the contents of which are hereby incorporated byreference.

Alternatively, the host organisms may be a bacterium, in particularstrains of Streptomyces and Bacillus, or E. coli. The transformation ofbacterial cells may be performed according to conventional methods, e.g.as described in T. Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor, 1982.

The screening of appropriate DNA sequences and construction of vectorsmay also be carried out by standard procedures, cf. T. Maniatis et al.,op. cit.

The medium used to cultivate the transformed host cells may be anyconventional medium suitable for growing the host cells in question. Theexpressed enzyme may conveniently be secreted into the culture mediumand may be recovered therefrom by well-known procedures includingseparating the cells from the medium by centrifugation or filtration,precipitating proteinaceous components of the medium by means of a saltsuch as ammonium sulphate, followed by chromatographic procedures suchas ion exchange chromatography, affinity chromatography, or the like.

When the bleaching enzyme employed in the invention is a peroxidase, H₂O₂ may be added at the beginning or during the process, e.g. in anamount of 0.001-5 mM, particularly 0.01-1 mM. When using Corpinusperoxidase 0.01-0.25 H₂ O₂ mM is preferred, and with B. pumilusperoxidase 0.1-1 mM H₂ O₂. When the bleaching enzyme employed in theprocess of the invention is a peroxidase, it may be desirable to utilizean enzymatic process for hydrogen peroxide formation. Thus, the processaccording to the invention may additionally comprise adding an enzymaticsystem (i.e. an enzyme and a substrate therefor) which is capable ofgenerating hydrogen peroxide at the beginning or during the washingand/or rinsing process.

One such category of hydrogen peroxide generating systems comprisesenzymes which are able to convert molecular oxygen and an organic orinorganic substrate into hydrogen peroxide and the oxidized substrate,respectively. These enzymes produce only low levels of hydrogenperoxide, but they may be employed to great advantage in the process ofthe invention as the presence of peroxidase ensures an efficientutilization of the hydrogen peroxide produced.

Preferred hydrogen peroxide-generating enzymes are those which act oncheap and readily available substrates which may conveniently beincluded into detergent compositions. An example of such a substrate isglucose which may be utilized for hydrogen peroxide production by meansof glucose oxidase. Other suitable oxidases are urate oxidase, galactoseoxidase, alcohol oxidases, amine oxidases, amino acid oxidase andcholesterol oxidase.

It has surprisingly been found that the addition of another oxidisablesubstrate (for the bleaching enzyme used in the process of theinvention) at the beginning or during the washing and/or rinsing processmay enhance the dye transfer inhibitory effect of the bleaching enzymeemployed. This is thought to be ascribable to the formation ofshort-lived radicals or other oxidised states of this substrate whichparticipate in the bleaching or other modification of the colouredsubstance. Examples of such oxidisable substrates are metal ions, e.g.Mn⁺⁺, halide ions, e.g. chloride or bromide ions, or organic compoundssuch as phenols, e.g. p-hydroxycinnamic acid or 2,4-dichlorophenol.Other examples of phenolic compounds which may be used for the presentpurpose are those given in M. Kato and S. Shimizu, Plant Cell Physiol.26(7), 1985, pp. 1291-1301 (cf. Table 1 in particular) or B. C. Saunderset al., opt. cit., p. 141 ff. The amount of oxidisable substrate to beadded is suitably between about 1 μM and 1 mM.

In the process of the invention, the bleaching enzyme Will typically beadded as a component of a detergent composition. As such, it may beincluded in the detergent composition in the form of a non-dustinggranulate, a liquid, in particular a stabilized liquid, or a protectedenzyme. Non-dusting granulates may be produced, e.g., as disclosed inU.S. Pat. Nos. 4,106,991 and 4,661,452 (both to Novo Industri A/S) andmay optionally be coated by methods known in the art. Liquid enzymepreparations may, for instance, be stabilized by adding a polyol such aspropylene glycol, a sugar or sugar alcohol, lactic acid or boric acidaccording to established methods. Other enzyme stabilizers are wellknown in the art. Protected enzymes may be prepared according to themethod disclosed in EP 238,216. The detergent composition may alsocomprise one or more substrates for the enzyme.

The detergent composition will additionally comprise surfactants whichmay be of the anionic, non-ionic, cationic, amphoteric, or zwitterionictype as well as mixtures of these surfactant classes. Typical examplesof anionic surfactants are linear alkyl benzene sulfonates (LAS), alphaolefin sulfonates (AOS), alcohol ethoxy sulfates (AES) and alkali metalsalts of natural fatty acids.

The detergent composition may further contain other detergentingredients known in the art as e.g. builders, anti-corrosion agents,sequestering agents, anti-soil redeposition agents, perfumes, enzymestabilizers, etc.

It is at present contemplated that, in the process of the invention, thebleaching enzyme may be added in an amount of 0.01-100 mg enzyme perliter of wash liquor.

The detergent composition may be formulated in any convenient form, e.g.as a powder or liquid. The enzyme may be stabilized in a liquiddetergent by inclusion of enzyme stabilizers as indicated above. Liquiddetergents may further include stabilized hydrogen peroxide precursors.Usually, the pH of a solution of the detergent composition of theinvention will be 7-12 and in some instances 7.0-10.5. Other detergentenzymes such as proteases, lipases or amylases may be included in thedetergent composition.

Hemopeptide

As noted above, one embodiment of the invention provides amicroperoxidase, i.e. a hemopeptide characterized by the amino acidsequence SEQ ID NO:3 having R linked to cysteine in position 1 and R'linked to histidine in position 5, wherein the sulphur atoms of the twocysteines are linked to the two vinyl groups of heme, and R and R' eachrepresents a peptide chain of 0-10 amino acids. These compounds can beprepared synthetically by methods known in the art, viz. by firstsynthesizing the peptide chain including the two cysteines and thenletting this peptide react with heme, or they can be producedgenetically by methods known in the art: a degenerate oligonucleotideprobe is synthesized (based on the known amino acid sequence) and usedto isolate the B. pumilus gene which encodes the precursor peptide chainof the microperoxidase. Modified microperoxidases are hereafter obtainedby site directed mutagenesis (ref. Molecular Cloning, A LaboratoryManual, Cold Spring Harbor, 1989, Ed., J. Sambrook, E. F. Fritsh and T.Maniatis, ISBN 0-87969-309-6).

Preferred embodiments include a hemopeptide characterized by the aminoacid sequence SEQ ID NO:5 having R' linked to histidine in position 9, ahemopeptide characterized by the amino acid sequence SEQ ID NO:6 havingR linked to cysteine in position 1 and a hemopeptide characterized bythe amino acid sequence SEQ ID NO:1; either of these may be shortened byone or more amino acids. These hemopeptides can be prepared bycultivation of a B. pumilus strain as described below.

A comparison of the sequences of hemopeptides of the invention with aknown microperoxidase (DE 3134526) prepared by hydrolysis of cytochromeC from animal or plant shows that the novel microbial microperoxidasehas very little homology with the known microperoxidase.

The prior art includes the hemopeptide having the amino acid sequenceSEQ ID NO:2 wherein the sulphur atoms of the two cysteines at positions5 and 8 are linked to the vinyl groups of heme.

A preferred embodiment of the present invention is the hemopeptidehaving the amino acid sequence SEQ ID NO:1 wherein the sulphur atoms ofthe two cysteines at positions 5 and 8 are linked to the vinyl groups ofheme.

Cultivation of B. pumilus

B. pumilus strains that can be used in the practice of the invention areatypical in their ability to produce peroxidase. Some B. pumilusstrains, including the type strain, have been found not to produceperoxidase.

Two peroxidase-producing strains are freely available with depositnumbers ATCC 12905 and NCIB 8600, and one strain (internal designation S197) has been deposited by the applicant under the terms of the BudapestTreaty; the deposit date was 23 Jul. 1990, and the deposit number is DSM6124.

ATCC indicates the American Type Culture Collection, 12301 ParklawnDrive, Rockville, Md. 20852, U.S.A., NCIB indicates the NationalCollection of Industrial and Marine Bacteria Ltd., Torry ResearchStation, P.O. Box 31, 135 Abbey Road, Aberdeen AB9 8DG, Scotland, andDSM indicates Deutsche Sammlung yon Mikroorganismen, Mascheroder Weg 1B,3300 Braunschweig, West Germany.

Peroxidase-producing B. pumilus strains can be cultivated under aerobicconditions in a nutrient medium containing assimilable carbon andnitrogen together with other essential nutrient. The medium can becomposed in accordance with principles known in the art.

During cultivation, the cells secrete peroxidase extracellularly, whilethey apparently produce catalase intracellularly. Extracellularproduction of the peroxidase is advantageous as it allows for highfermentation yield and simple recovery. Catalase is undesired for mostapplications of peroxidase, so the recovery of peroxidase preferablyincludes separation of cell mass from the cell broth while avoiding celllysis, e.g. by filtration or centrifugation.

The resulting cell-free culture broth contains a mixture of hemopeptideswith peroxidase activity, mainly with molecular weight in the range800-2500. This can be used as such, optionally after concentration e.g.by evaporation or ultrafiltration. If desired, the various hemopeptidescan be separated and purified to the desired degree by conventionalmethods, e.g. by column chromatography.

pH and temperature dependence

A crude peroxidase preparation of the invention (FIG. 1) and a similarpurified preparation (FIG. 2) have slightly different pH optimum, butboth are in the range 7-9.

The thermostability of peroxidase preparations of the invention (FIG. 3and 4) is significantly better than a prior art microbial peroxidase(FIG. 5). A crude preparation according to the invention (FIG. 3) ismore thermostable than a similar purified preparation. Both preparationsof the invention retain more than 40% residual activity after 30 minutesincubation at 80° C. at pH 7.

Hydrogen peroxidase dependence

A peroxidase preparation of the invention (FIG. 6) shows increasingreaction rate up to 20 mM H₂ O₂ (the highest tested). For comparison, aprior art microbial peroxidase shows decreasing reaction rate at H₂ O₂concentrations above 0.1 mM (FIG. 7).

Bleaching of lignin-containing material

Due to its good stability at high temperature and high H₂ O₂concentration, the peroxidase preparation of the invention is wellsuited for bleaching of lignin-containing materials, such as pulp forpaper production or waste water from pulp manufacturing, together withhydrogen peroxide, e.g. as described in Ser. No. 88/0673, U.S. Pat. No.4,623,465 or JP-A 2-31887. Typical conditions are pH 7-10, 25°-70° C.,10-300 mM H₂ O₂ and a dosage of 10-100 NOPA/g dry matter. Hydrogenperoxide may be added as such or formed in situ, e.g. by incorporationof a precursor, such as a perborate or percarbonate, or of an enzymaticsystem capable of generating hydrogen peroxide, e.g. an oxidase and asubstrate therefor (such as glucose and glucose oxidase).

Other uses of peroxidase

Use of the peroxidase preparation of the invention is particularlyadvantageous at high temperature and/or high H₂ O₂ concentration and atalkaline pH. As an example, it can be incorporated into a laundrydetergent together with a hydrogen peroxide precursor (such as sodiumperborate or percarbonate) to improve the bleaching of stains accordingto WO 89/09813.

As another example, the peroxidase preparation can be used to inhibitthe transfer of a textile dye from a dyed fabric to another fabric whenthe fabrics are washed and/or rinsed together in a wash liquor, byadding it together with a hydrogen peroxide precursor to the wash liquorin which the fabrics are washed and/or rinsed.

Peroxidase assay (NOPA)

Peroxidase activity is measured at 2 mM H₂ O₂. The following are mixedin a 30° C. thermostated 1 ml quartz cuvette:

200 μl 1 mM 4-aminoantipyrine (4-AA, Sigma No. A-4382, 0.2 mg/ml)

200 μl N-ethyl-N-sulphobutyl-m-toluidine-Na (ESBT, 5.86 mg/ml)

200 μl 0.5 M phosphate buffer, pH 7.0

200 μl enzyme sample, diluted to 0.02-0.10 NOPA/ml

200 μl 10 mM hydrogen peroxide is added, and the absorbance at 550 nm isfollowed for 1 minute. The activity is expressed in units denoted NOPA,calculated as the increase in absorbance within the first minute afteraddition of H₂ O₂ multiplied by the dilution. The enzyme sample shouldbe diluted so that the increase in absorbance per minute is within thelimits 0.02 to 0.10.

BRIEF DESCRIPTION OF DRAWINGS

FIGS. 1 and 2 show the pH-activity curve of a crude and a purifiedperoxidase preparation, respectively, according to the invention,measured by the NOPA method, but varying the pH as shown.

FIGS. 3 and 4 illustrate the thermostability of a crude and a purifiedperoxidase of the invention, respectively. For comparison, FIG. 5 showsthe thermostability of a prior-art peroxidase preparation (fromCoprinus). The measurements were made by incubating at the indicatedtime and temperature, then immediately diluting a sample 10 times in 0.1M phosphate buffer pH 7 at 25° C., and measuring peroxidase activity(NOPA).

FIGS. 6 and 7 illustrate reaction at various H₂ O₂ concentrations withperoxidase of the invention and prior-art peroxidase (Coprinus),respectively. Oxidation of ESBT+4-AA was followed by measuring theabsorbance at 550 nm.

FIGS. 1 and 3 were made using cell-free culture broth prepared as inExample 13. FIGS. 2, 4, and 6 were made using pool III from Example 14.

FIGS. 8-11 show chromatograms from purification of peroxidase of theinvention. Details are given in Example 14.

EXAMPLES

Dyes were purchased from Aldrich Chemicals. Peroxycarboxylic acidreferences were synthesized according to W. E. Parker, C. Ricciuti, C.L. Ogg and D. Swern, J. Am. Chem. Soc., 77, 4037 (1955). Spectra wererecorded on a Hewlett Packard 8451 diode array spectrophotometer. Thesamples were scanned over the wavelength range 200 to 800 nm for oneminute (spectra recorded every 6 sec). CMP is used below as anabbreviation for peroxidase derived form Coprinus macrorhizus (obtainedfrom Chemical Dynamics). H₂ O₂ is used synonymously with hydrogenperoxide. 2,4-DCP and PCA are used as abbreviations of2,4-dichlorophenol and p-coumaric acid.

EXAMPLE 1

Bleaching of Congo Red in solution

To a solution of Congo Red (0.058 mM, 42 mg/l (dye content 93%, givingan initial abosrbance at 486 nm of 2.0) in phosphate buffer pH 7 (0.1 M)was added as bleaching agent either 2 mM H₂ O₂, 1 mM peroxyoctanoicacid, or 2.5 mg/l CMP+0.25 mM H₂ O₂. The experiments were performed at25° C. in 1 cm quartz cells containing 1 ml. As listed below, only theperoxidase system gave any bleaching effect (monitored as observedchange in absorbance at 486 nm in one minute).

    ______________________________________                                        Bleaching system  Delta absorbance in 1 min                                   ______________________________________                                        2 mM H.sub.2 O.sub.2                                                                            0.00                                                        1 mM Peroxyoctanoic acid                                                                        0.00                                                        2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2                                                          0.18                                                        ______________________________________                                    

EXAMPLE 2

Bleach acceleration by phenolic compounds

Experiments were performed according to, example 1, except that theaccelerating effect of adding various phenolic compounds as anadditional substrate along with the peroxidase and H₂ O₂ was examined.2,4-DCP and PCA were added at a level of only 5 μM (0.82 mg/l in bothcases).

    ______________________________________                                                                 Delta absorbance                                     Bleaching system         in 1 min                                             ______________________________________                                        2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2                                                                 0.18                                                 2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2 + 5 μM 2,4-DCP                                               0.74                                                 2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2 + 5 μM PCA                                                   0.28                                                 ______________________________________                                    

EXAMPLE 3

Bleaching of Acid Blue 45 in solution

Experiments were performed according to example 1 only using a solutionof Acid Blue 45 (0.058 mM, 68 mg/l (dye content ca 40 %), giving aninitial absorbance at 594 nm of 1.0). Bleaching was measured as changein absorbance at 594 nm.

    ______________________________________                                        Bleaching system  Delta absorbance in 1 min                                   ______________________________________                                        2 mM H.sub.2 O.sub.2                                                                            0.00                                                        1 mM Peroxyoctanoic acid                                                                        0.00                                                        2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2                                                          0.42                                                        ______________________________________                                    

EXAMPLE 4

Bleach acceleration by phenolic compounds

Experiments were performed as described in example 2 except for usingAcid Blue 45 as described in example 3.

    ______________________________________                                                                Delta absorbance                                      Bleaching system        in 1 min                                              ______________________________________                                        2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2                                                                0.42                                                  2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2 + 5 μM 2,4-DCP                                              0.69                                                  2.5 mg/1 CMP + 0.25 mM H.sub.2 O.sub.2 + 5 μM PCA                                                  0.98                                                  ______________________________________                                    

EXAMPLE 5

Solutions of Congo Red and Acid Blue 45 prepared, according to example 1and 3, were treated with laccase (100 mg/l, crude enzyme preparation,derived from Mycoliophtora thermophile, available from Novo Nordisk as aspecial preparation, SP 315. Further information is available uponrequest). The difference in absorbance relative to a solution withoutenzyme added was measured after an incubation time of 16 hours.

    ______________________________________                                                 Difference in absorbance after 16 hr.                                Bleaching agent                                                                          Congo Red (486 nm)                                                                          Acid Blue 45 (594 nm)                                ______________________________________                                        0.1 g/l laccase                                                                          0.29          0.09                                                 ______________________________________                                    

EXAMPLE 6

Dye adsorption to textiles

In order to demonstrate that the effects seen in the above solutionexperiments are reflected on textiles present in such solutions,experiments were carried out in which clean cotton swatches wereimmersed in solutions of model textile dyes.

In one such experiment, the clean swatches were immersed in 0.058 mM and0.012 mM solutions, respectively, of the dye Acid Blue 45 in 50 mMphosphate buffer (pH 7.0, 25° C.) and agitated for 60 min. The phosphatebuffer was freshly prepared from water of a hardness equivalent to 1.6mM Ca²⁺. The swatch load was approx. 11 g cotton cloth/l.

Afterwards the swatches were rinsed in tap water and air-dried in thedark on a clean towel overnight. The remission at 600 nm (absorptionregion for blue substances) was measured on a Datacolor Elrephometer2000.

The results of three treatments within the above prescriptions were asfollows:

    ______________________________________                                                 Remission at 600 nm (%)                                                       Swatches retrieved                                                                        Swatches retrieved                                                from 0.058 mM Acid                                                                        from 0.012 mM Acid                                                Blue 45 solution                                                                          Blue 45 solution                                         ______________________________________                                        1.  Reference  60            80                                                   (buffer only)                                                             2.  0.2 mM H.sub.2 O.sub.2                                                                   58            79                                               3.  H.sub.2 O.sub.2 as in 2 +                                                                74            90                                                   20 mg/l CMP                                                               ______________________________________                                    

Higher remission numbers here correspond to less blue color. Thus, thedye deposition on the clean swatches is considerably less in thesolutions with peroxidase present.

EXAMPLE 7

Dye Adsorption to textiles

In another experiment, the procedure of example 6 was repeated in everydetail, except that the dye in the solutions was Congo Red (at the samemM levels). Here, visual inspection of the resulting swatchesunequivocally demonstrated the effect of the peroxidase: treatments 1and 2 gave indistinguishably and heavily red-colored swatches, whereasonly a faint yellowish color was seen on the swatches from treatment 3.

EXAMPLE 8

Dye adsorption to textiles

In this experiment, a particular type of test swatch was added fordemonstrating dye adsorption effects. Each swatch consisted of 6 stripsof textile, each 1.5 cm by 5 cm, sown together; the 6 textile brandswere triacetate, bleached cotton, nylon, polyester, orlon, and viscoserayon.

The model washing liquor was a phosphate buffer prepared as in example 6with 0.6 g/l linear alkylbenzenesulfonate added as a surfactant. Two 7cm by 7 cm clean cotton swatches and one of the above multiswatches(also clean) were immersed in 1 litre of the washing liquor, with CongoRed added to a level of 0.012 mM, in each of two Terg-o-tometer beakers.In beaker 1, the bleaching system consisted of H₂ O₂ at a level of 2 mM,in beaker 2, 20 mg of CMP was further added. A wash of 30 min at 40° C.with 60 rotations/min was performed, after which the swatches wererinsed in tap water and dried as above (example 6). This time, Huntercolor difference readings were obtained for the multiswatches asfollows:

    ______________________________________                                                    Hunter color difference readings                                              Beaker 1                                                                              Beaker 2                                                              (only H.sub.2 O.sub.2)                                                                (H.sub.2 O.sub.2 + CMP)                                   ______________________________________                                        Triacetate     7.5       2.0                                                  Cotton        69.9      35.0                                                  Nylon         57.2      23.4                                                  Polyester     16.0       5.0                                                  Orlon         27.4       9.8                                                  Viscose       69.7      30.7                                                  ______________________________________                                    

(A value of 0 here indicates no change in color from the clean swatchand increasing numbers correspond to a visual impression of deepercolor.)

Thus, the conclusion from example 6 is also valid here for all thetextile brands studied.

EXAMPLE 9

Dye transfer from textile to textile

Swatches dyed with Congo Red as a model dye for azo textile dyes wasprepared by immersing clean cotton swatches in a bath of Congo Red andsodium sulfate in demineralized water and keeping them there during agradual heating to 90° C., ending with addition of further sodiumsulfate and a period of a constant temperature of 90° C. After beingdyed the swatches were rinsed in cold tap water and dried overnightbetween layers of gauze.

In the present experiment, washing was carried out in threeTerg-o-tometer beakers under the same general conditions as in example8. The contents of the beakers were:

Beaker 1: Only phosphate buffer with LAS (as in example 8)

Beaker 2: Buffer+LAS+2 mM H₂ O₂

Beaker 3: As 2 with 20 mg/l CMP added

In each beaker was introduced 2 Congo Red swatches, 7 cm by 7 cm, andone clean multiswatch (see example 8). After washing and drying as inexample 8, the Hunter readings of the multiswatches were as follows:

    ______________________________________                                                Beaker 1   Beaker 2 Beaker 3                                          ______________________________________                                        Triacetate                                                                               3.4          3.4      2.8                                          Cotton    45.7         45.3     36.6                                          Nylon     41.6         40.9     35.6                                          Polyester  7.9          7.4      6.7                                          Orlon     14.7         15.0     11.2                                          Viscose   45.1         44.6     36.3                                          ______________________________________                                    

Thus, the swatches in beaker 1 suffer a substantial dye transfer whichis not remedied by hydrogen peroxide alone, but reduced significantly bythe peroxidase treatment.

The red swatches from the three beakers had essentially identicalreadings, showing that the peroxidase treatment does not change thedyeing any more than the other treatments.

EXAMPLE 10

Dye adsorption to textiles

For the purpose of studying the peroxidase effect in a more realisticwashing environment, a powder detergent was composed as follows:

    ______________________________________                                        Component              w/w % active material                                  ______________________________________                                        Sodium carbonate       22                                                     Sodium diphosphate     17                                                     Sodium silicate        7                                                      Sodium triphosphate    5                                                      Sodium perborate monohydrate                                                                         4                                                      Sodium nonanoyloxybenzenesulfonate                                                                   5                                                      Sodium linear alkylbenzenesulfonate                                                                  9                                                      Sodium alkyl sulfate   4                                                      Various minor components: alcohol ethoxylate,                                                        each < 1                                               diethylenetriamine pentaacetate, polyacrylate,                                polyethylene glycol, protease, optical                                        brightener                                                                    Sodium sulfate and other miscellaneous                                                               balance                                                ______________________________________                                    

This detergent was used at a level of 2 g/l in water of a hardnessequivalent to 1.6 mM Ca²⁺ to produce a washing liquor in which pH wasadjusted to 8.5. In this washing liquor, Congo Red was dissolved to alevel of 0.012 mM. Beaker 1 was the reference (detergent+Congo Red); inbeaker 2, CMP was added to a level of 20 mg/l. In both beakers, twoclean cotton swatches and one clean multiswatch were added as in example8. All other conditions were as in example 8 and the Hunter data for themultiswatches after the wash were as follows:

    ______________________________________                                                     Beaker 1                                                                             Beaker 2                                                  ______________________________________                                        Triacetate      4.0     1.1                                                   Cotton         62.5     2.3                                                   Nylon          48.0     1.1                                                   Polyester       4.0     0.4                                                   Orlon          18.4     1.2                                                   Viscose        66.3     1.3                                                   ______________________________________                                    

Once again, the peroxidase clearly reduces--here almost eliminates--theamount of color deposited on the swatches.

EXAMPLE 11

Dye transfer from textile to textile

In this example, the detergent solution from example 9 was used in aTerg-o-tometer trial where two of the Congo Red-dyed swatches describedabove (example 9) were washed together with one clean multiswatch ineach of two beakers. Beaker 1 contained just 1 litre of detergentsolution, beaker 2 additionally contained 20 mg/l of CMP. The remainingconditions were as in example 8. The swatches, after retrieval from thewashing liquor, rinsing and drying as above, showed the following Huntercolor difference data:

    ______________________________________                                                     Beaker 1                                                                             Beaker 2                                                  ______________________________________                                        Triacetate      2.3      1.1                                                  Cotton         47.0     13.1                                                  Nylon          36.0     11.3                                                  Polyester       2.1      1.1                                                  Orlon           6.5      2.7                                                  Viscose        48.7     10.6                                                  ______________________________________                                    

A considerable transfer of dye was thus observed in beaker 1, and thiswas significantly reduced by adding the peroxidase to the washingliquor.

Again, the dyed swatches were checked also, and no color difference wasseen between the two treatments.

EXAMPLE 12

Bleaching of dyestuffs in solution

Peroxidase activity: In this example, the peroxidase activity ismeasured as follows. The following are mixed in a 30° C. thermostated 1ml quartz cuvette:

200 μl 1 mM 4-aminoantipyrine (Sigma No. A-4382, 0.2 mg/ml)

200 μl N-ethyl-N-sulphobutyl-m-toluidin-Na (ESBT, 5.86 mg/ml)

200 μl 0.5 M phosphate buffer, pH 7.0

200 μl enzyme sample, diluted to 0.02-0.10 NOPA/ml

200 μl 10 mM hydrogen peroxide is added, and the absorbance at 550 nm isfollowed for 1 minute. The activity (in NOPA units) is calculated as theincrease in absorbance within the first minute after addition of H₂ O₂multiplied by the dilution. The enzyme sample should be diluted so thatthe increase in absorbance per minute is within the limits 0.02 to 0.10.

Peroxidase production from Bacillus pumilus: Media were prepared asfollows (ingredients in g/l):

    ______________________________________                                                      TY*3                                                            ______________________________________                                        Trypticase, BBL g/l                                                                           60                                                            Yeast Extract, Difco g/l                                                                      15                                                            FeSO.sub.4 *7H.sub.2 O g/l                                                                     0.025                                                        MnSO.sub.4 *4H.sub.2 O g/l                                                                      0.0026                                                      MgSO.sub.4 *7H.sub.2 O g/l                                                                     0.045                                                        pH              7.3                                                                           (Adjusted with KOH)                                           ______________________________________                                    

The medium was autoclaved at 121° C. for 45 minutes.

    ______________________________________                                                        Agar3                                                         ______________________________________                                        Pepton Bacto g/l  6                                                           Pepticase g/l     4                                                           Yeast Extract, Difco g/l                                                                        3                                                           Meat Extract g/l    1.5                                                       Glucose           1                                                           pH                  7.3                                                       Agar (from Merck) 20                                                                            (added last)                                                ______________________________________                                    

The agar was autoclaved at 121° C. for 45 minutes.

Inoculum agar: 10 Agar3 slants were inoculated with a freeze-driedperoxidase-producing strain of B. pumilus sign and incubated at 30° C.for 24 hours.

Inoculum media: Two 500 ml Shake flasks containing 100 ml TY*3 mediawere incoluated with one Agar3 slant and incubated at 30° C. and 250 rpmfor 24 hours.

Peroxidase production: 50 Shake flasks containing 100 ml of TY*3 wereincoluated each with 2 ml of inoculum described above. Then 2.5 ml of asterile 40% (w/w) Glucose in water was added to each shake flask. Theshake flasks were incubated at 30° C. for 48 hrs and then harvested. Thefinal peroxidase activity was 1 NOPA/ml.

3250 ml culture broth were filtered through a Seitz Supra 100filterplate and secondly through a Supra 50 plate to obtain a clearfiltrate with an activity of 1.29 NOPA/ml.

Bleaching of dyes in solution: The above clear filtrate from B. pumilus(BPP) was tested. The dyestuffs tested were Direct Blue 1 (C.I. #24410,product of Keystone Aniline), Acid Red 151 (C.I. #26900, product ofSandoz), Procion Blue H ERD (product of ICI) and Procion Blue EXL(product of ICI).

A reaction solution was prepared, containing 50 mM sodium phosphate, 0.3NOPA/ml of peroxidase, dyestuff (as indicated below) corresponding to anabsorption maximum (in the visible range) of 0.025-0.035, and 0.25 mM atroom temperature at the pH indicated below. After addition of H₂ O₂(added last), a spectral scan was made every minute for 12 minutes.Below, the change in absorbance at the wavelength of maximum absorptionis listed.

    ______________________________________                                                            Absorbance change                                                                            Wave-                                      Dyestuff   pH       immediately/after 12 min                                                                     length                                     ______________________________________                                        Acid Red 151                                                                             7.0      0.030/0.632    513 nm                                                9.0      0.033/0.033    513 --                                                10.5     0.027/0.030    513 --                                     Direct Blue 1                                                                            7.0      0.024/0.625    597 nm                                                9.0      0.022/0.026    597 --                                                10.5     0.009/0.023    597 --                                     Procion Blue                                                                             7.0      0.022/0.021    617 nm                                     H ERD      9.0      0.009/0.022    617 --                                                10.5     0.001/0.010    617 --                                     Procion Blue                                                                             9.0      0.021/0.026    626 nm                                     H EXL      10.5     0.016/0.025    626 --                                     ______________________________________                                    

When the two values of the absorbance change are close, the bleaching ispractically instantaneous. Generally, bleaching over the entire visiblerange follows the above trends at the absorbance maximum.

In all cases, use of 0.25 mM H₂ O₂ without enzyme left the dyeunchanged.

EXAMPLE 13

Production of peroxidase from Bacillus pumilus

Media were prepared as follows (ingredients in g/l):

    ______________________________________                                                      TY*3                                                            ______________________________________                                        Trypticase, BBL g/l                                                                           60                                                            Yeast Extract, Difco g/l                                                                      15                                                            FeSO.sub.4 *7H.sub.2 O g/l                                                                     0.025                                                        MnSO.sub.4 *4H.sub.2 O g/l                                                                      0.0026                                                      MgSO.sub.4 *7H.sub.2 O g/l                                                                     0.045                                                        pH              7.3                                                                           (Adjusted with KOH)                                           ______________________________________                                    

The medium was autoclaved at 121° C. for 45 minutes.

    ______________________________________                                                        Agar3                                                         ______________________________________                                        Pepton Bacto g/l  6                                                           Pepticase g/l     4                                                           Yeast Extract, Difco g/l                                                                        3                                                           Meat Extract g/l    1.5                                                       Glucose           1                                                           pH                  7.3                                                       Agar (from Merck) 20                                                                            (added last)                                                ______________________________________                                    

The agar was autoclaved at 121° C. for 45 minutes.

Inoculum agar: 10 Agar3 slants were inoculated with 1 freeze driedBacillus pumilus Strain no S197 and incubated at 30° C. for 24 hrs.

Inoculum media: Two 500 ml Shake flasks containing 100 ml TY*3 mediawere inoculated with one Agar3 slant and incubated at 30° C. and 250 rpmfor 24 hrs.

Peroxidase production: 50 Shake flasks containing 100 ml of TY*3 wereinoculated each with 2 ml of inoculum described above. Then 2.5 ml of asterile 40% (w/w) glucose in water was added to each shake flask. Theshake flasks were incubated at 30° C. for 48 hrs and then harvested. Thefinal peroxidase activity was 1 NOPA/ml (NOPA measured at 20 mM H₂ O₂).

EXAMPLE 14

Purification of peroxidase from B. pumilus

3250 ml of the culture broth obtained in Example 13 was filtered througha Seitz Supra 100 filter plate and secondly through a Supra 50 plate toobtain a clear filtrate with an activity of 1.29 NOPA/ml.

To 900 ml of the filtrate was added 70 g Amberlite XAD16 hydrophobicresin and the mixture was stirred overnight at 4° C. The resin wasisolated by decantation and washed with 10% v/v ethanol. Peroxidase wasextracted from the resin with 200 ml 50% v/v ethanol which wasevaporated to 72 ml with an activity of 13.8 NOPA/ml (yield: 86%).

The evaporated peroxidase extract was applied to a DEAE Sepharose FFcolumn (5×8 cm) equilibrated with 20 mM phosphate pH 7. The column waswashed with more than one volume of buffer containing 0.1 M NaCl andperoxidase was eluted with buffer containing 0.3 M NaCl. Fractions of 10ml were collected and pooled in three pools according to protein (A280)and activity profiles (FIG. 8):

    ______________________________________                                        Fraction no.                                                                              ml      A280   A404   NOPA/ml                                                                              Yield                                ______________________________________                                        Pool A                                                                              23-26     40      8.14 1.37   2.33   9.3%                               Pool B                                                                              27-30     40      6.41 1.05   2.17   8.7%                               Pool C                                                                              36-42     40      8.90 1.84   6.89   27.7%                              Total yield: 45.7%                                                            ______________________________________                                    

A 90 ml sample with 7.34 NOPA/ml of pool C was added 1.6 M ammoniumsulphate to a conductivity of 108 mS (pH 7.05) and applied on anOctyl-Sepharose column (5×11) cm) equilibrated with 0.8 M ammoniumsulphate pH 7. Unbound material was eluted with 0.8 M ammonium sulphateand peroxidase activity was then eluted with a linear gradient ofammonium sulphate from 0.8 to 0 molar (pure water).

Fractions of 10 ml were collected and pooled in three pools according tothe peroxidase activity (FIG. 9):

    ______________________________________                                        Fraction no.                                                                              ml      A280   A398   NOPA/ml                                                                              Yield                                ______________________________________                                        Pool I                                                                              84-92     90      0.26 0.19   1.47   20.0%                              Pool II                                                                             94-96     30      0.58 0.37   2.09   9.5%                               Pool III                                                                             97-100   40      0.55 0.30   1.57   9.5%                               Total yield: 39%                                                              ______________________________________                                    

A sample of Pool III was concentrated by ultrafiltration on a 50 mlAmicon stirred cell with a DDS GS90PP membrane. A 7.5 ml concentratedsample with an activity of 131.2 NOPA/ml was applied to a Sephacryl-S200column (2.5×85 cm) equilibrated with 12.5 mM phosphate buffer pH 7.0 andeluted with the same buffer at a flow rate of 0.9 ml/min. Fractions of9.8 ml were collected and pooled according to peroxidase activity andspectral properties (Rz-value: A398/A280) (FIG. 10):

    ______________________________________                                        Fraction no.                                                                              ml      A280   A398   NOPA/ml                                                                              Yield                                ______________________________________                                        Pool A                                                                              47-50     38      0.47 1.22   11.2   43.3%                              Pool B                                                                              45        9.8     0.73 0.69    6.76   6.7%                              Pool C                                                                              46        9.8     1.16 1.65   15.97  15.8%                              Total yield: 65.8%                                                            ______________________________________                                    

The mass spectrum of Pool IA show one major peak at Mw 2214.0/2228.1 andtwo minor peaks at Mw 972.8 and 1768.1, respectively. The amino acidsequence of the major component was shown to be SEQ ID NO:4 where eachXaa is believed to be cysteine through which a heme group (responsiblefor the A398 absorbance) can be bound, i.e., the amino acid sequence SEQID NO:1.

A sample of pool II was concentrated by ultrafiltration on a 50 mlAmicon stirred cell with a DDS GS90PP membrane. A 10 ml concentratedsample with an activity of 143.8 NOPA/ml was applied to a AcA 202 column(2.5×85 cm) equilibrated with 12.5 mM phosphate buffer pH 7.0 and elutedwith the same buffer at a flow rate of 0.4 ml/min. Fractions of 5 mlwere collected and monitored for A280, A398 and peroxidase activity(FIG. 11).

This AcA 202 gel is claimed by the manufacturer to separate molecules inthe range from 1 kD to 15 kD. From the chromatogram it is seen that awide range of relatively low Mw substance was eluted and all showing thesame peroxidase activity relative to the A398 (Soret) absorbance.

EXAMPLE 15

Bleaching of kraft pulp

The peroxidase used in this example was the permeate fromultrafiltration of the supernatant from alcohol precipitation of acell-free culture broth (obtained essentially as in Example 13).

1 g hardwood (birch) kraft pulp, delignified by oxygen, was blended in250 ml water adjusted to pH 8.0-8.2 with NaOH. H₂ O₂ was added to aconcentration of 1% (0.3 M), and the above peroxidase preparation wasadded to a dosage of 0.32 NOPA/ml. The mixture was incubated at 40° C.for 2 hours. Then the pulp was collected on a Buchner funnel and washedwith sufficient water to obtain a clear filtrate. The pulp was pressedand dried, and the ISO brightness of the obtained sheet was measured. Areference was made in the same way without the enzyme.

Invention: 57.2% ISO brightness

Reference: 55.6% ISO brightness

EXAMPLE 16

Bleaching of CTMP pulp

The experiment of Example 15 was repeated at 30° C. with CTMP softwood(pine) pulp.

Invention: 59.7% ISO brightness

Reference: 56.1% ISO brightness

EXAMPLE 17

Bleaching of dyes in solution

The effect of the peroxidase preparation of the invention in bleaching adissolved textile dye was tested. The dyestuffs tested were Direct Blue1 (C.I. #24410, product of Keystone Aniline), Acid Red 151 (C.I. #26900,product of Sandoz), Procion Blue HERD (product of ICI) and Procion BlueH EXL (product of ICI). The peroxidase preparation was cell-free culturebroth prepared essentially as in Example 13.

A reaction solution was prepared, containing 50 mM sodium phosphate, 0.3NOPA/ml of peroxidase, dyestuff (as indicated below) corresponding to anabsorption maximum (in the visible range) of 0.025-0.035, and 0.25 mM H₂O₂ at room temperature at the pH indicated below. After addition of H₂O₂ (added last), a spectral scan was made every minute for 12 minutes.Below, the change in absorbance at the wavelength of maximum absorptionis listed.

    ______________________________________                                                            Absorbance change                                                                            Wave-                                      Dyestuff   pH       immediately/after 12 min                                                                     length                                     ______________________________________                                        Acid Red 151                                                                             7.0      0.030/0.032    513 nm                                                9.0      0.033/0.033    513 --                                                10.5     0.027/0.030    513 --                                     Direct Blue 1                                                                            7.0      0.024/0.025    597 nm                                                9.0      0.022/0.026    597 --                                                10.5     0.009/0.023    597 --                                     Procion Blue                                                                             7.0      0.022/0.021    617 nm                                     H ERD      9.0      0.009/0.022    617 --                                                10.5     0.001/0.010    617 --                                     Procion Blue                                                                             9.0      0.021/0.026    626 nm                                     H EXL      10.5     0.016/0.025    626 --                                     ______________________________________                                    

When the two values of the absorbance change are close, the bleaching ispractically instantaneous. Generally, bleaching over the entire visiblerange follows the above trends at the absorbance maximum.

In all cases, use of 0.25 mM H₂ O₂ without enzyme left the dyeunchanged.

This example illustrates the potential usefulness of the peroxidasepreparation of the invention in preventing surplus dye fromstrongly-coloured fabric from being redeposited during washing.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 6                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5..8                                                            (D) OTHER INFORMATION: /note="Heme group attached to Cys                      in positions 5 and 8"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GlnGluGlnThrCysIleSerCysHisGlyAspAsnMetGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5..8                                                            (D) OTHER INFORMATION: /note="Heme group attached to Cys                      in positions 5 and 8"                                                         (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       PheValGlnLysCysAlaGlnCysHisThrValGluLysGly                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note="Heme group attached to Cys                      in positions 1 and 4"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="R attached to Cys in                            position 1, wherein R is a peptide chain of 0-10 amino                        acids"                                                                        (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5                                                               (D) OTHER INFORMATION: /note="R'attached to His in                            position 5, wherein R'is a peptide chain of 0-10 amino                        acids"                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       CysIleSerCysHis                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 14 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5..8                                                            (D) OTHER INFORMATION: /note="Xaa in positions 5 and 8                        represents any amino acid"                                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       GlnGluGlnThrXaaIleSerXaaHisGlyAspAsnMetGln                                    1510                                                                          (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 5..8                                                            (D) OTHER INFORMATION: /note="Heme group attached to Cys                      in positions 5 and 8"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 9                                                               (D) OTHER INFORMATION: /note="R'attached to His in                            position 9, wherein R'is a peptide chain of 0-10 amino                        acids"                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlnGluGlnThrCysIleSerCysHis                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1..4                                                            (D) OTHER INFORMATION: /note="Heme group attached to Cys                      in positions 1 and 4"                                                         (ix) FEATURE:                                                                 (A) NAME/KEY: Modified-site                                                   (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note="R attached to Cys in                            position 1, wherein R is a peptide chain of 0-10 amino                        acids"                                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CysIleSerCysHisGlyAspAsnMetGln                                                1510                                                                          __________________________________________________________________________

We claim:
 1. A process for bleaching a textile dye in a solution ordispersion, the process comprising incorporating an effective amount ofan enzyme exhibiting oxidase activity on phenolic compounds into thesolution or dispersion.